The broad, long-term objective of the proposed research is to characterize antigen processing and presentation pathways in dendritic cells (DC), and how these relate to their function in initiating and regulating immune responses. The proposed research is intended to determine the location within DC where peptide and protein-derived antigens are loaded onto class II MHC proteins, and how this is regulated by dendritic cell maturation;to determine the molecular basis for the lack of loading of empty class II MHC proteins present in immature DC;to evaluate possible functional roles for empty class II MHC molecules;and to determine structural characteristics of the peptide-free open conformation of HLA-DR1. These goals will be achieved using cellular and biochemical assays to follow the occupancy and peptide binding activity of class II MHC proteins in antigen presenting cells in different developmental states, and using cellular and immunological assays to follow the functional outcomes of antigen presentation. A novel fluorometric probe of peptide binding has been developed for this purpose. PROJECT NARRATIVE: Antigen presentation by dendritic cells plays a key role in the process by which the immune system recognizes and responds to pathogens. A detailed understanding of the basic cellular processes responsible for antigen presentation will be important to guide therapeutic approaches to regulating inappropriate immune responses as in autoimmunity and allergy, and for efforts to develop new vaccines and to improve existing vaccines against infectious agents and cancer.